Astrocytes are not merely passive members of the circuit, but that they may also be directly involved with neuronal computation. Indeed, astrocytes are well suited to a potential widespread role in the circuit: they tile the CNS with near- complet coverage, are connected into an extensive syncytium via gap junctions, and processes from a single astrocyte can contact up to tens of thousands of synapses. In fact, we have recently shown how even the stimulation of single astrocyte can control circuit-wide neuronal synchronizations in the cortex. Tools to study astrocytes are significantly less developed than those available to investigate neuronal function and, consequently, most research on astrocytic information processing has been limited and incomplete. Astrocyte researchers need methods to selectively measure and manipulate the function of astrocytes in vivo. Two-photon excitation, which penetrates living tissue while affording single-cell resolution, combined with compatible optochemical or optogenetic probes, could be an ideal technical platform for future astrocyte research. We propose to develop novel two-photon and computational tools to provide subcellular and circuit-level analysis of astrocytic function in neural circuits in vivo, using a nvel astrocyte-specific Brainbow mouse line. These tools include two- photon optogenetic and caged compounds, methods to image groups of astrocytes in 3D, and computational algorithms to reveal interactions of astrocytes with neurons in the circuit. With this award, we will provide the astroglia community with a wide range of optical and computational tools that can be readily adapted for use in vivo. This work will enable the investigation of the roles of astrocytes in shaping neuronal activity in the cortical microcircuit and beyond, and thus could have a fundamental impact on how we view neuro-glia processing, as well as on solidifying the role of an overlooked circuit constituent-the astrocyte-in cortical synchrony and computation. Finally, this work could introduce a novel potential means to assay or control neuronal function for potential therapeutic purposes: through specific activation of astrocytes.